Coral snakes, known for their red, yellow, and black color patterns, deliver a venomous bite that can be fatal. Their neurotoxic venom inhibits the muscles' ability to receive signals from nerves, causing paralysis and death by suffocation. Current antivenoms are expensive to produce, cover limited coral snake species, cause adverse side effects, and contain few therapeutically active antibodies.
In a recent study, researchers at the Technical University of Denmark and Universidad Nacional Autónoma de México developed cross-neutralizing nanobodies against neurotoxic phospholipase A2 and alpha-neurotoxin, two key toxins in coral snake venoms, as reported in Nature Communications (1). Their approach offers a significant advantage over current alternatives, including neutralizing all medically relevant toxins cost-effectively and ensuring a high therapeutic antibody content in the recombinant products.
Neutralizing snake venom is challenging due to the diversity of proteins within a single snake's venom and variations across different species. “We had wondered for a long time whether you could get a good phospholipase A2-neutralizing antibody and a good [alpha-neurotoxin] neutralizing antibody and whether those could protect against the entirety of the venom,” said Joseph Jardine, an immunologist at The Scripps Research Institute who was not involved with the study. “This [work] shows that taking out those two problematic toxins is enough to protect.”
Existing antivenom technology, in use for nearly 130 years, involves immunizing a large animal, typically a horse, with snake venom, and then isolating antibodies from the horse's plasma. While these antibodies effectively neutralize snake venom, they can cause adverse reactions in humans and need to be administered in high doses since a single injection contains just a handful of neutralizing antibodies.
To create a more affordable antivenom that requires smaller doses, the study authors used alpacas and llamas to produce special antibodies called nanobodies. They then made collections of bacteriophages that display these nanobodies on their surfaces. The researchers inserted many different DNA sequences into the bacteriophages, each coding for a different nanobody. They then mixed these bacteriophages with a toxin, allowing the nanobodies that can bind to the toxin to be identified. Each bound nanobody is attached to a virus that contains its DNA, making it easy to find out the nanobody's exact genetic sequence. Once the sequence is known, scientists can produce large amounts of the nanobody synthetically.
“[Nanobodies] are only a tenth of the size of a regular antibody, are cheap to produce at a large scale, are very stable, and bind just as well as a big antibody,” said Andreas Laustsen, antivenom and toxicology researcher at the Technical University of Denmark and study coauthor. “A small size means you will need fewer grams to neutralize antivenom, which is key for making cheaper therapies.”
[Nanobodies] are only a tenth of the size of a regular antibody, are cheap to produce in large scale, are very stable, and bind just as well as a big antibody. A small size means you will need fewer grams to neutralize antivenom, which is key for making cheaper therapies.
- Andreas Laustsen, Technical University of Denmark
In in vitro experiments, the nanobodies showed high affinity binding and cross-reactivity to the classical coral snake neurotoxins phospholipase A2 and alpha-neurotoxin and to other snakes that produce similar toxins such as cobras and mambas.
In mice, the anti-phospholipase A2 and anti-alpha-neurotoxin nanobodies effectively neutralized the lethality of both phospholipase A2 and alpha-neurotoxin, respectively. Laustsen’s group prepared oligoclonal mixtures of one nanobody neutralizing phospholipase A2 and one nanobody neutralizing alpha-neurotoxin to treat mice exposed to the full venom which contains both neurotoxins. They found comparable neutralization capacity to Coralmyn, a traditional antivenom, while neither nanobody alone prevented lethality or prolonged survival in envenomed mice. The oligoclonal mixtures also showed broader species coverage, effectively neutralizing toxins from the venoms of two different coral snake species.
In future experiments, Laustsen hopes to test his newly developed nanobodies on larger animals such as sheep or pigs, with the goal of making better antivenoms available to people. “This is exciting work, because it's leveraging cutting edge biotechnology to go after an incredibly old problem,” Jardine said. “This has the potential to be superior to what's currently out there.”
Even if Laustsen’s approach fails, he believes that the nanobody concept has broad applicability beyond snakebites. “Making mixtures of broadly neutralizing antibodies would be relevant in infectious diseases, and even autoimmune diseases and cancers where you need to target multiple things at the same time,” he said.
Reference
- Benard-Valle, M. et al. In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model. Nat Commun 15, 4310 (2024).
